Differential extracellular vesicle concentration and their biomarker expression of integrin αv/β5, EpCAM, and glypican-1 in pancreatic cancer models

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

than 1% of the total EV concentration for all measurements.Immunoblot analysis performed on the three EV samples revealed the presence of the EV marker proteins CD9, CD63, and flotillin-1 (Fig. 1B, Supplementary Fig. 1) 29 .All samples exhibited robust and consistent expression of flotillin-1 and CD63, with slight variations observed in the levels of CD9.This data validates the quality of EVs extracted via different methods.Notably, the total EV concentration varied depending on the isolation methods.Both UC and TEIR-based methods yielded approximately 1.5 times more EVs compared to the SEC-based method (Fig. 1C).Nevertheless, the peak value of the size distribution, representing the modal size of EVs, remained consistent across all three isolation techniques (Fig. 1A,D).Additionally, scanning electron microscopy and atomic force microscopy images (Fig. 1A) confirmed the consistent modal size of the EV observed in the NTA observation.

Quantification methods influence EV concentration
The quantification of EV size and concentration is commonly performed using the NTA method in various research fields related to EVs.However, an alternative EV quantitation method is the colorimetric enzyme assaying-based technique (EXOCET), which offers the advantage of assessing EV concentration using standard laboratory equipment, instead of a stand-alone NTA instrument.In this study, we examined both NTA and EXOCET to quantify EV concentrations isolated from the plasma of healthy, PNET, and PDAC patients using the UC method.When compared to NTA measurements, the EXOCET method provided EV counts that were up to three as high in all tested plasma samples (Fig. 2).Additionally, the EXOCET measurements exhibited a higher degree of variability in repeated measurements for the same EV samples compared to the NTA method (Fig. 2A).Nevertheless, there were no statistical differences observed in the quantification results between the two methods.More importantly, the overall trend in total counts across the four different EV samples remained consistent between both measurement techniques (Fig. 2B).

Elevated EV concentration in pancreatic cancer with unchanged modal size
To compare the size and concentration of EVs between pancreatic cancer and non-cancerous conditions, we isolated EVs using the UC method from various sources including cell lines (HPNE, BxPC-3, PANC-1), mouse models (wild-type and KPC), and human plasma (healthy, PNET, and PDAC) and charactered them using the NTA method.Figure 3A depicts the normalized size distribution of each EV sample and compares the size probabilities between PDAC and non-cancerous EVs.Statistical analysis revealed no significant differences in size distribution across all EV samples.Moreover, a consistent modal size of approximately 125 nm was observed (Fig. 3B), regardless of the EV source.
Unlike the uniformity in the EV modal size, a notable contrast was observed in the overall EV concentration when comparing pancreatic cancer and non-cancerous EV samples (Fig. 3C).Specifically, the EV secretion by PANC-1 cells significantly exceeded that by HPNE cells.While the comparison of EV quantities between BxPC-3 and HPNE cells did not reach statistical significance (P = 0.11), the mean total EV count in BxPC-3 was consistently at least double that of HPNE cells.This pattern of elevated total EV quantities in pancreatic cancer was also observed in the mouse model (KPC vs. wild-type) and patient plasma (PDAC vs. healthy control) samples.Remarkably, the total number of EVs isolated from PDAC patient plasma was statistically distinguishable from both healthy and PNET cases.

Unique EV biomarker expression profiles in pancreatic cancer
To assess the potential of EVs as biomarkers of pancreatic cancer, we investigated the relative protein expression levels of four EV biomarkers (ITGα v , ITGβ models, and patient plasma (Fig. 4).Western blot analysis revealed significantly higher expression levels of all four biomarkers in EVs from the PDAC cell lines (BxPC-3 and PANC-1) compared to the normal HPNE cell line (Fig. 4A).Specifically, ITGα v , ITGβ 5 , GPC-1, and EpCAM exhibited statistically distinguishable expression differences between the BxPC-3 and HPNE cell lines.In the comparison between the PANC-1 and HPNE cell lines, ITGα v , ITGβ 5 , and EpCAM expression showed significant differences with a mild increase in GPC-1 expression observed in PANC-1 cells.Similarly, in mouse models, EVs from the KPC mouse model displayed significantly higher expression levels of ITGα v , GPC-1, and EpCAM compared to those from the wild-type mouse model (Fig. 4B, Supplementary Fig. 2).However, no significant difference was observed in the expression level of ITGβ 5 .Finally, we analyzed the protein expression of the four EV biomarkers in plasma EVs from healthy and PDAC patients (Fig. 4C, Supplementary Fig. 2).Notably, their relative expression significantly increased in PDAC EVs compared to healthy controls.This observation, consistent across cell lines, mouse models, and human samples, suggests their potential as diagnostic EV markers for PDAC.

Discussion
We conducted a comprehensive evaluation of two quantification methods, NTA and EXOCET, and determined that both methods consistently provide reproducible and reliable results when analyzing samples of EVs from cancer and non-cancerous sources.Interestingly, our analysis revealed that the size and concentration distribution of EVs isolated through three different isolation techniques were also nearly identical, implying that neither the specific method of EV isolation nor the quantification technique significantly impacts the relative EV quantification or the size distribution.Furthermore, our investigation showed a significant increase in the concentration of EVs isolated from PDAC cell lines, mouse models, and patient plasma when compared to their respective control groups.Markedly, the size distribution of these EVs remained consistent across all EV sources.These compelling observations suggest that   www.nature.com/scientificreports/pancreatic tumors may actively promote the secretion of EVs.Additionally, we observed significantly elevated expression levels of EV biomarkers ITGα v , ITGβ 5 , GPC-1, and EpCAM in EVs isolated from PDAC compared to non-cancerous conditions.This finding suggests a strong correlation between PDAC and the expression of these EV biomarkers.
Our results highlight the potential of both the relative EV concentration and the presence of these four EV protein signatures as specific biomarkers for the identification and prediction of PDAC.However, it is important to note that the total EV count and the quantity of EV biomarkers include contributions from both normal and tumor-derived EVs in PDAC patient plasma.Therefore, it is reasonable to assume that the differences in EV count and EV biomarker expression are predominantly driven by tumor-derived EVs.This enables us to assess the absolute increase in biomarker expression per tumor-derived EV in comparison to normal EVs.
By utilizing data from the fold-increase of the total EV count in PDAC (N c ) and the fold-increase of relative biomarker expression levels in PDAC (N b ), the fold-increase in biomarkers on individual tumor-derived EVs (X) compared to normal EVs can be estimated by X = (N c N b − 1)/(N c − 1), which represents the lower limit of the biomarker fold-increase in tumor-derived EVs.Given that both N c and N b for PDAC patients are approximately 2, we estimate that the fold-increase in biomarkers on individual tumor-derived EVs X is 3, indicating a minimum threefold increase in the biomarkers on these EVs.
In summary, EVs and their distinct biomarkers show promise for distinguishing PDAC from healthy conditions, highlighting their potential in both PDAC detection and advancing our understanding of PDAC biology.

Animal studies
The disease progression and genotyping for the KPC mice were previously described 31 .KPC mice in C57BL/6J background were established as described previously at the NDSU animal facility 32 .WT C57BL/6J (000664) was purchased from Jackson Laboratory.The animals were kept in a pathogen-free, humidity (50-70%) and temperature (22-25 °C) controlled environment with a 12 h light/dark cycle.Fresh food and water will be provided ad libitum.KPC mice were monitored for tumor development using Vevo 3100 ultrasound.Blood samples were collected via retro-orbital puncture (100-400 ml, terminal bleeding), followed by euthanization for tumor collection.The average age of 3 KPC mice used the sample collection was four months.All animal experiments were reviewed and approved by the Institute of Animal Care and Use Committee at North Dakota State University.All experiments were conducted in accordance with ARRIVE guidelines.The conduct and reporting of the described experiments adhere to the "Guide for the Care and Use of Laboratory Animals" and the "U.S.Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training".

Patient plasma
Plasma samples from patients with pathologically-verified pancreatic neuroendocrine tumors (PNET) and pancreatic ductal adenocarcinomas (PDAC), and healthy donors, who have no history of pancreatic disease or cancer, were collected at the University of Pittsburgh Medical Center as part of the Institutional Review Board approved Pancreatic Adenocarcinoma Gene Environment Risk (PAGER)-a prospective cohort study (IRB no.STUDY19070256).Written informed consent was obtained from patients to allow blood samples to be used for research.All experiments were performed in accordance with the IRB guidelines and regulations.The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University of Pittsburgh Medical Center.

Ultracentrifuge EV isolation (UC)
Once cells were grown sub-confluent, cells were washed with PBS and the medium was changed to contain exosome-depleted FBS (Gibco™, A2720801) at 10%.The cells were then cultured for an additional 48 h.The culture supernatant was subjected to sequential centrifugation steps at 800g for 5 min and 2000g for 10 min, then filtered with 0.2 mm filters.The filtrate was subjected to ultracentrifugation at 28,000 RPM using an SW 28 Ti swinging bucket overnight to collect EVs.The supernatant was aspirated, and the pellet was resuspended in PBS.EVs collected from 30 ml of cell culture supernatant were resuspended in 200 µl of PBS.For both mouse and human plasma samples, 0.5 ml of plasma samples were centrifuged at 2,000 RPM at 4 °C for 30 min.The plasma sample was then mixed with PBS in a 1:20 ratio and filtered through a 0.2 μm filter.Filtrate was then centrifuged at 40,000 RRM overnight using Beckman SW 41 Ti.After ultracentrifugation, the supernatant was decanted, and the pellet was resuspended in 500 μl PBS.The purified EVs were then further analyzed.

EV reagent isolation (TEIR)
A total exosome isolation reagent kit (4,478,360, Invitrogen) was used to isolate EVs according to manufacturer instructions.Briefly, 0.5 ml of plasma was centrifuged at 2000g for 30 min to remove all debris.Clarified plasma and provided reagent were mixed in a 5:1 ratio and incubated at 4 °C for 30 min.Precipitated EVs were then centrifuged for 10 min at 10,000g, and the supernatant was discarded.The EV pellet was then resuspended in PBS, and the purified EVs were further analyzed.www.nature.com/scientificreports/

Size exclusion chromatography EV isolation (SEC)
A qEV isolation column (qEV/35 nm, IZON Science) was used to isolate EVs according to manufacturer instructions.Briefly, 0.5 ml of plasma samples were centrifuged at 1500g for 10 min, then the supernatant was transferred to a new tube and centrifuged again at 10,000g for 10 min.Columns were equilibrated, clarified plasma was added to the column, and flow-through containing EVs was collected according to manufacturer instructions.

Nanoparticle tracking analysis
The size and concentration distribution of EVs were determined by nanoparticle tracking analysis (NTA) using the NanoSight NS300 system (Malvern Panalytical Ltd, UK).The EV samples were diluted to 1000-fold in PBS for NTA measurements.The samples were infused with the syringe pump at a constant speed of 20 into the microfluidic flow cell equipped with a 532 nm laser and a high-sensitivity scientific CMOS camera.At least three videos per sample were recorded with a camera level of 11-13 for 30 s at 25 °C.All data were analyzed using NTA software (version 3.4) with a detection threshold of 4-6.

EV colorimetric assay
An EXOCET exosome quantitation kit (EXOCET96A-1, SBI) was used to quantify the EV samples using the necessary reagents included in the kit according to manufacturer instructions.Briefly, isolated EVs were incubated in the provided lysis buffer to liberate associated proteins.Samples were then vortexed briefly and centrifuged at 1500g for 5 min.The supernatant was transferred to 96 well plates and incubated with reaction buffer for 15 min at room temperature.The reaction plate was read using xMark™ Microplate Absorbance Spectrophotometer immediately at 405 nm.Results were quantified by calculating the standard curve and plotting the sample readings on the standard curve.

EV imaging
A 10-20 μL droplet of EVs in PBS was placed on a SiO 2 substrate for 60 min at 4 °C.The samples were then washed with PBS and dried under nitrogen flow.Imaging of the samples was performed using atomic force microscopy (NTEGRA, NT-MDT) 33 and scanning electron microscopy (JSM-7600F, JEOL).

Statistical analysis
All data were expressed as mean ± standard deviation and analyzed using the Prism 8 (GraphPad software).The statistical significance was determined using Student's t-test and analysis of variance (ANOVA) followed by a suitable post-hoc test.The p-values lower than 0.05 were considered statistically significant.

Figure 1 .
Figure 1.Comparison of three EV isolation methods on human plasma.(A) The size and concentration distribution of EVs isolated using UC, TEIR, and SEC methods, as determined by the NTA technique.The color shade represents the standard deviation (n = 3 technical replicates).The inset shows the light scattering image of the EVs.The top and bottom panels show SEM and AFM images of the EVs, respectively.(B) Immunoblot of CD9, CD63, and flotillin-1 EV marker proteins.50 μg of EV protein extract was loaded per lane.(C) EV concentration of the identical PDAC patient plasma (n = 3 technical replicates), and (D) their modal EV size.Data are presented as mean ± standard deviation.

Figure 3 .Figure 4 .
Figure 3. EV size and concentration in PDAC cell lines, mouse models, and patients.(A) The normalized size distribution of EVs isolated from each source, as determined by the NTA technique.The color shade represents the standard deviation (n = 3 per group).(B)The modal size and (C) concentration for each source.The statistical analysis was performed using either Student's t-test or one-way ANOVA followed by Tukey's post-hoc test (*P < 0.05, **P < 0.01; n = 3 per group).Data are presented as mean ± standard deviation.The statistics of the modal particle size among all EVs showed no difference (not significant). https://doi.org/10.1038/s41598-024-65209-8

5 , GPC-1, and EpCAM) in EVs isolated from PDAC cell lines, mouse A B NTA EXOCET 0 5×10 11 1×10 12 1.5×10 12
NTA EXOCETFigure 2. Comparison of two EV quantification methods.(A)TheEV concentration of the identical PDAC patient, as determined by NTA and EXOCET techniques (n = 5 technical replicates).The statistics of the concentration between the two methods showed no statistical difference (not significant).(B) EV concentration of four different EV samples as determined by the two quantification techniques (Student's t-test, *P < 0.05, **P < 0.01; n = 3 technical replicates per group).Data are presented as mean ± standard deviation.The statistics of the concentration between the two methods for PNET and PDAC1 showed no statistical difference (not significant) unless otherwise noted.